Vandoorne K, Raz T, Sapoznik S, Biton IE, Garbow JR, Neeman M. In Vivo Preclinical Imaging of Developmental Biology. In: Small Animal Imaging. ; 2017.Abstract

Embryonic development, the generation of a living organism from a fertilized egg, poses some of the most intriguing challenges of biological research. Aberrations in development can arise from genetic alterations in the fetus, which can vary in the degree of penetration, and can also result from direct and indirect pathological processes affecting the fetus or the mother. Impaired fetal development is a major cause of premature morbidity and mortality. Dynamic imaging of the live fetus provides an important tool for elucidating the normal and pathological developmental changes occurring during pregnancy. In particular, as part of efforts for functional mapping of the genome using genetically modified animals, detailed analysis of fetal development in laboratory animals is central in elucidating the function of genes and the impact of alteration in gene expression. Moreover, imaging biomarkers developed in the context of basic biological research could provide the foundations for future prenatal clinical imaging. In this chapter, we will review recent developments in the use of noninvasive imaging for longitudinal monitoring of live embryos in small laboratory animals, with particular focus on in utero imaging of fetal development in the mouse.

Klainbart S, Slon A, Kelmer E, Bdolah-Abram T, Raz T, Segev G, Aroch I, Tal S. Global hemostasis in healthy bitches during pregnancy and at different estrous cycle stages: Evaluation of routine hemostatic tests and thromboelastometry. Theriogenology. 2017.Abstract

This study assessed the global hemostasis (including prothrombin time [PT], activated partial thromboplastin time [aPTT], antithrombin activity [ATA], fibrinogen and d-Dimer concentrations, platelet count, plateletcrit and thromboelastometry) in healthy pregnant bitches, comparing the results with those of healthy bitches at different estrous cycle stages, and assessed whether hemostatic changes during pregnancy are associated with serum progesterone concentration or the presence of fetuses in utero. The results show that pregnant bitches have higher fibrinogen concentration, platelet count and platelatecrit, and that fibrin and global clot formations occur faster than in non-pregnant bitches at different estrous cycle stages. Additionally, clot strength was higher in pregnant bitches than in non-pregnant ones. There were no differences in PT, ATA, and D-dimer concentration between all study groups. The aPTT was significantly shorter in bitches at the fourth and last pregnancy weeks, compared to the anestrus group, and shorter in both the fourth and last pregnancy weeks groups, compared to diestrus group. These results all support a hypercoagulable state in healthy pregnant bitches, unassociated with progesterone concentration.

Gunther I, Raz T, Zor YE, Bachowski Y, Klement E. Feeders of Free-Roaming Cats: Personal Characteristics, Feeding Practices, and Data on Cat Health and Welfare in an Urban Setting of Israel. Frontiers in Veterinary Science. 2016.Abstract

Cat feeders serve as an important source of available food for free-roaming cats (FRC) and can play a central role in providing data on FRC distribution, welfare, and health. Data on cat feeder personalities as well as a better understanding of their feeding practices offer relevance for decision making concerning FRC population control strategies. The current study surveyed 222 FRC feeders who responded to a municipal trap-neuter-return (TNR) campaign in an Israeli central urban setting. The aim of the study was to describe their personal characteristics, feeding practices and the FRC populations they feed. Feeders were divided into four groups according to the number of cats they claimed to feed per day (group 1: fed up to five cats; group 2: fed six to 10 cats; group 3: fed 11-20 cats; group 4: fed ≥21 cats). Most feeders were women (81%), with a median age of 58 years (range 18-81). The feeders reported an overall feeding of 3,337 cats in 342 different feeding locations. Feeders of group 4 comprised of 15.31% (n=34) of all feeders, but fed 56% (n=1869) of the FRC in 37.42% (n=128) of the feeding locations. 'Heavy' feeders (groups 3 and 4) reported that they traveled significantly longer distances in order to feed the cats. Commercial dry food consisted of 90% of the food they provided, with 66% of them feeding once a day, with less food per cat per day than the other feeder groups. Interestingly, 'heavy' feeders were usually singles, had on average fewer siblings, a clear preference for owning cats as pets and lived in lower income neighborhoods. According to the feeders' reports on the FRC populations they fed, 69.7% (2325/3337) cats were neutered and 11.8% (395/3337) were kittens. In addition, they reported that 1.6% (54/3337) of the cats were limping, 2% (67/3337) suffered from a systemic disease, 4% (135/3337) had skin lesions, and 3.9% (130/3337) were suffering from a chronic disability. Abundance of kittens and morbidity rate were significantly and negatively associated with neutering rate. These findings are in accordance with the suggestion that neutering may potentially improve cat welfare by reducing morbidity.

Oreff GL, Dahan R, Tatz AJ, Raz T, Britzi M, Kelmer G. The Effect of Perfusate Volume on Amikacin Concentration in the Metacarpophalangeal Joint Following Cephalic Regional Limb Perfusion in Standing Horses: Regional Limb Perfusion of Amikacin in Standing Horses. Veterinary Surgery . 2016.Abstract

Objective: To determine the influence of 3 perfusate volumes on amikacin concentration in the metacarpophalangeal joint following cephalic regional limb perfusion (RLP) in standing horses. Animals: Seven healthy horses. Methods: Three perfusate volumes (100, 60, and 30 mL), containing 2 grams of amikacin, were tested during intravenous RLP at the cephalic vein, placing the tourniquet at mid antebrachium, in standing sedated horses. Synovial fluid was collected from the metacarpophalangeal joint before perfusion and at 30 and 120 minutes after perfusion. Serum samples were taken from the jugular vein at the same time points. Samples were analyzed for amikacin concentrations and a repeated measures ANOVA, followed by least squares difference pairwise comparisons to identify differences in amikacin concentration across perfusate volumes. Differences were considered significant at P<.05. Results: The mean amikacin concentration in synovial fluid at 30 minutes after perfusion was significantly higher following perfusate volume of 100 mL (579 μg/mL), compared to volumes of 60 mL (227 μg/mL) or 30 mL (282 μg/mL) (P<.05). When a threshold of 160 μg/mL was used, more horses reached the synovial therapeutic threshold following perfusate volume of 100 mL (100%), than horses receiving 60 mL (43%) and 30 mL (57%) at 30 minutes after injection. Conclusion: The use of 100 mL volume for RLP at the cephalic vein in standing horses resulted in higher concentration of amikacin in the synovial fluid and is recommended for use in clinical cases.

Gunther I, Raz T, Berke O, Klement E. Nuisances and welfare of free-roaming cats in urban settings and their association with cat reproduction. Preventive Veterinary Medicine . 2015.Abstract

Free roaming cats (FRC) are highly abundant in cities around the world. Increasing populations of these cats might result in impairment of cat welfare and cause nuisances and public health risks. In order to study the seasonal dynamics of FRC populations and its association with events of cat welfare impairment and nuisances, we analyzed a database of FRC-associated citizens' telephone complaint events, which were registered in five cities in Israel (total human population of 1.42 million residents) during the years 2007-2011. These complaint events were classified to the following six categories: cat's carcasses, kittens, parturition, aggressive behavior toward people, invasion to human facilities, and cat injuries and distress. Overall, 87,764 complaint events associated with these categories were registered in the five cities during the study period (123.2 complaint events per 10,000 citizens per year). Length of daylight was moderately correlated with the rate of complaints on kittens in the same month (r=0.64) and parturition in the previous month (r=0.54) (P<0.001). Both kitten and parturition-related complaints showed a prominent seasonal pattern, peaking in April and May, respectively, and declining gradually until November. 'Kittens' or 'parturition' were explicitly mentioned in 38%, 39% and 19%, respectively, of the complaints regarding cat aggressiveness toward people, cat invasion to human facilities and cat injuries and distress. In most of the cities the rate of citizen complaints regarding carcasses, aggression, invasion and injuries were still significantly correlated with rate of complaints regarding kittens after omission of these joint complaints and remained significant after controlling for seasonality. These findings imply an association of cat welfare impairment and nuisances with FRC reproduction intensity. The current study revealed the high rate of nuisances and potential public health hazards related to FRC, as well as the impairment of cat welfare, which might be merely 'the tip of the iceberg' of the real welfare situation of these cats. Further studies should examine the effectiveness of FRC population control strategies for the reduction of the rate of nuisances and public health risks related to FRC, as well as for improving their welfare. Copyright © 2015 Elsevier B.V. All rights reserved.

Cohen Y, Dafni H, Avni R, Fellus L, Bochner F, Rotkopf R, Raz T, Benjamin LE, Walsh K, Neeman M. Genetic and Pharmacological Modulation of Akt1 for Improving Ovarian Graft Revascularization in a Mouse Model. Biology of Reproduction. 2015.Abstract

Ovarian tissue cryopreservation and transplantation is one of a few available treatments for fertility preservation in women diagnosed with cancer. Rapid revascularization is essential for reducing hypoxic damage after grafting and protecting the primordial follicles reserve. Using a mouse model of heterotopic ovarian graft transplantation, we have delineated the role of endothelial Akt1 expression using longitudinal MRI follow-up to quantify angiogenic response. Endothelial Akt1 activation in ovarian grafts promoted angiogenesis to support the graft during post-transplantation hypoxic period. Similarly, simvastatin therapy activated Akt1 at the transplantation site and improved the revascularization and vascular support of ovarian grafts. These results serve as an important first step towards pharmacological intervention to improve revascularization of ovarian grafts and restoration of fertility in cancer survivors. The pro angiogenic effects reported here may extend beyond improving ovarian graft reception in fertility preservation and could potentially be used for different organ or tissue transplantation.

Cohen A, Tadmor A, Raz T, Meterani N, Addadi Y, Nevo N, Solomonov I, Sagi I, Mor G, Neeman M, et al. Ovarian Dendritic Cells Act as a Double-Edged Pro-Ovulatory and Anti-Inflammatory Sword.  Molecular Endocrinology . 2014.Abstract

Ovulation and inflammation share common attributes, including immune cells invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and FACS analyses, we demonstrate that CD11c-positive, F4/80-negative-cells, representing DCs, are recruited to the ovary under gonadotropins regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of cumulus oocytes complex, release of ovum from ovarian follicle, formation of a functional corpus luteum and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DCs transplantation following conditional ablation of CD11c-positive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by upregulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. On top of discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classical immunologic role, which is relevant also to other biological systems.

Solomon E, Avni R, Hadas R, Raz T, Garbow JR, Bendel P, Frydman L, Neeman M. Major mouse placental compartments revealed by diffusion-weighted MRI, contrast-enhanced MRI, and fluorescence imaging. Proceedings of the National Academy of Sciences . 2014.Abstract

Mammalian models, and mouse studies in particular, play a central role in our understanding of placental development. Magnetic resonance imaging (MRI) could be a valuable tool to further these studies, providing both structural and functional information. As fluid dynamics throughout the placenta are driven by a variety of flow and diffusion processes, diffusion-weighted MRI could enhance our understanding of the exchange properties of maternal and fetal blood pools-and thereby of placental function. These studies, however, have so far been hindered by the small sizes, the unavoidable motions, and the challenging air/water/fat heterogeneities, associated with mouse placental environments. The present study demonstrates that emerging methods based on the spatiotemporal encoding (SPEN) of the MRI information can robustly overcome these obstacles. Using SPEN MRI in combination with albumin-based contrast agents, we analyzed the diffusion behavior of developing placentas in a cohort of mice. These studies successfully discriminated the maternal from the fetal blood flows; the two orders of magnitude differences measured in these fluids' apparent diffusion coefficients suggest a nearly free diffusion behavior for the former and a strong flow-based component for the latter. An intermediate behavior was observed by these methods for a third compartment that, based on maternal albumin endocytosis, was associated with trophoblastic cells in the interphase labyrinth. Structural features associated with these dynamic measurements were consistent with independent intravital and ex vivo fluorescence microscopy studies and are discussed within the context of the anatomy of developing mouse placentas.

Cohen B, Ron E, Raz T, Addadi Y, Ben-Dor S, Eilam R, Yaniv K, Neeman M. Bach1 is a novel modulator of angiogenesis and lymphangiogenesis. Angiogenesis . 2014.
Raz T, Avni R, Neeman M. Multimodal Imaging of the Mouse Placenta. In: The Guide to Investigation of Mouse Pregnancy. ; 2014.Abstract

Multimodal imaging is an important part of the study of placenta structure and function, as well as embryonic development, particularly in transgenic mice. The development of novel imaging techniques can contribute significantly to enhance our understanding of placental structure, exchange within the placenta, as well as the architecture and function of the maternal and fetal vasculature system, all of which are critical in the evaluation of the dynamic relationships between the mother, placenta, and fetus during pregnancy. Recent progress of a number of imaging approaches is described herein, including non-invasive MRI, ultrasound imaging, fluorescence microscopy, and photoacoustic imaging. Applications of these techniques are used to monitor the details of blood-flow patterns in the uterine arteries, to measure placental perfusion, and to characterize angiogenesis and vascular permeability, providing insight into placental and fetal pathologies. All of these findings eventually provide a better appreciation for the unique properties of mammalian development and reproduction.

Avni R, Raz T, Neeman M. MRI, Intra-vital, and Ex-vivo Fluorescence Microscopy of the Mouse Uterine Vasculature and Placenta. In: The Guide to Investigation of Mouse Pregnancy. ; 2014.Abstract

MRI and fluorescence microscopy provide complementary information on the structure and function of placental blood vessels. Due to the highly effective fetal-maternal barrier, dynamic contrast enhanced MRI using macromolecular contrast media probes exclusively the maternal blood flow in the placenta, providing information on the rate of flow into the placenta. Arterial spin labeling follows the motion of water across the fetal-maternal barrier, and thus this approach can provide information on perfusion. Arterial spin labeling achieved by tagging water passing through the uterine versus the ovarian arterial input, provide directional information for the two blood supplies, and can also help in non invasive positioning of the embryos along the uterine horns. Fluorescence intravital and ex vivo imaging provides for faster dynamic analysis and validation of maternal blood volume respectively.

Kelmer G, Raz T, Berlin D, Steinman A, Tatz AJ. Standing open-flank approach for removal of enlarged pathologic ovaries in mares. 2013.
Vandoorne K, Vandsburger M, Raz T, Shalev M, Weisinger K, Biton IE, Brumfeld V, Raanan C, Nevo N, Eilam R, et al. Chronic Akt1 Deficiency Attenuates Adverse Remodeling and Enhances Angiogenesis After Myocardial Infarction. Circulation Cardiovascular Imaging. 2013.Abstract

-Akt1 is a key signaling molecule in multiple cell types including endothelial cells. Accordingly Akt1 was proposed as a therapeutic target for ischemic injury in the context of myocardial infarction (MI). The aim of this study was to use multi-modal in vivo imaging to investigate the impact of systemic Akt1 deficiency on cardiac function and angiogenesis before and after MI. -In vivo cardiac magnetic resonance imaging (MRI) was performed before and at day 1, 8, 15 and 29-30 after MI induction for wildtype, heterozygous, and Akt1 deficient mice. Non-infarcted hearts were imaged using ex vivo stereomicroscopy and μCT. Histological examination was performed for non-infarcted hearts and for hearts at days 8 and 29-30 post-MI. MRI revealed mildly decreased baseline cardiac function in Akt1 null mice, while ex vivo stereomicroscopy and μCT revealed substantially reduced coronary macrovasculature. After MI, Akt1(-/-) mice demonstrated significantly attenuated ventricular remodeling and a smaller decrease in ejection fraction. At 8 days after MI a larger functional capillary network at the remote and border zone, accompanied by reduced scar extension, preserved cardiac function, and enhanced border zone wall thickening, were observed in Akt1(-/-) mice when compared to littermate controls. -Using multi-modal imaging to probe the role of Akt1 in cardiac function and remodeling after MI, this study revealed reduced adverse remodeling in Akt1 deficient mice after MI. Augmented myocardial angiogenesis coupled with a more functional myocardial capillary network may facilitate revascularization and therefore be responsible for preservation of infarcted myocardium.

Cohen Y, Dafni H, Avni R, Raz T, Biton IE, Hemmings B, Neeman M. In search of signaling pathways critical for ovarian graft reception: Akt1 is essential for long-term survival of ovarian grafts. Fertility and sterility. 2013.Abstract

To explore the role of Akt1, a principle modulator of angiogenesis, in ovarian graft reception and to investigate whether Akt1 deficiency can alter ovarian graft reception. Experimental mouse model. Research institute. Donors: Akt1 knockout (Akt1(-/-)) and wild types (Akt1(+/+)) mice. Recipients: CD-1 nude immune deficient female mice. Ovaries from Akt1(-/-) and Akt1(+/+) mice transplanted in the biceps femoris muscle of immunocompromised CD-1 mice, and ovarian graft viability, perfusion, and revascularization explored in vivo by magnetic resonance imaging (MRI). Vascular density and permeability of newly formed graft blood vessels quantified by dynamic contrast-enhanced MRI 7, 14, 30, and 60 days after grafting as indicators for angiogenesis and reestablishment of blood perfusion. The Akt1(-/-) ovarian grafts showed a gradual decrease in angiogenic response with time after transplantation, ultimately leading to complete or near-complete graft destruction coinciding with massive follicular loss. Sixty days after transplantation, the mean blood volume fraction (fBV) and vessel permeability (PS) were statistically significantly lower in Akt1(-/-) transplants compared with Akt1(+/+). Akt1 is essential for ovarian graft reception. However, surprisingly the impact of Akt1 deficiency was most profound not in the early stages of angiogenesis but rather in long-term survival of the graft.